The osmoregulation system in Saccharomyces cerevisiae offers a unique opportunity in which to explore key features of signal transduction pathways that are prevalent in both prokaryotic and eukaryotic cells. The initial steps of this signaling pathway involve multiple histidine-to-aspartate phosphoryl transfer events involving three proteins, SLN1, YPD1, and SSK1, that are related to bacterial "two-component" signal transducers. This phosphorelay system, in turn, regulates a downstream mitogen- activated protein (MAP) kinase cascade. The research proposed here focuses on understanding structure/function relationships that dictate molecular interactions and hence phosphoryl transfer. X-ray crystallographic studies coupled with mutagenesis experiments of the histidine-containing phosphorelay protein YPD1 will yield insight regarding surface(s) of YPD1 that interact with the homologous domains associated with SLN1 and SSK1. The response regulator protein, SSK1, represents a novel regulator of a MAP kinase cascade. Thus, by initiating structural studies of SSK1, it is anticipated that we will uncover unique and important information regarding the role of phosphorylation in regulating SSK1 function and the molecular surface(s) of SSK1 that are available for interaction with the downstream proteins of the MAP kinase cascade. This study will have broad based implications relevant to the many two-component signal transduction systems in bacteria and MAP kinase-dependent signaling pathways in higher eukaryotes. Moreover, because related MAP kinase cascades function in pathways that control cell growth and differentiation and since histidine-to-aspartate phosphoryl transfer is essential for the tumor-suppressive activity of human Nm23 protein, the research proposed here will likely provide insights into the etiology of cancer. Overall, the fundamental objective of this research is to better understand protein phosphorylation as a universal form of cellular regulation.